Abstract Damage-associated molecular pattern molecules (DAMPs) are intracellular molecules that are actively secreted or passively released by damaged cells. Once released, DAMPs promote inflammation and tissue repair. The high-mobility box group 1 (HMGB1) protein is a prototypic and highly conserved DAMP which acts as a pro-inflammatory cytokine by activating pattern recognition receptors (PRRs) including Toll-Like Receptor 2 (TLR2), TLR4, and the Receptor for Advanced Glycation End Products (RAGE). Evidence suggests that in response to persistent tissue injury, HMGB1 acts as a key initiating molecule of innate immunity, orchestrating inflammation and tissue remodeling. Preliminary data implicate HMGB1, TLR2 and TLR4 in the development of pulmonary arterial hypertension (PAH) in animal and tissue culture models. The central hypothesis of this proposal is that HMGB1, via activation of PRRs, is a key mediator of innate immunity and contributes to endothelial activation and vascular remodeling in PAH. Two specific aims are proposed to test this hypothesis. Specific aim 1 will focus on characterizing the expression and location of HMGB1 and PRRs in idiopathic PAH (IPAH) and non-PAH patient samples. We will use immunostaining to localize HMGB1, TLR2, TLR4, and RAGE expression to specific lung structures and cell types, focusing on areas of vascular remodeling. We will quantify the lung-specific expression of HMGB1, TLR2, TLR4. We will use ELISA and Luminex assays to quantify cytokines and chemokines related to activation of the innate immune system as well as HMGB1 in plasma and lung from IPAH and non-PAH patients. These data will correlate the location- specific DAMP release, PRR-expression pattern and level and pro-inflammatory cytokine production that are central to our main hypothesis. Specific aim 2 will focus on elucidating a role for HMGB1 and PRRs in vascular remodeling and endothelial activation. We will: A) Measure mRNA and protein expression of TLR2, TLR4, and Rage in pulmonary artery endothelial cells (PAEC) and pulmonary artery smooth muscle cells (PASMC) from IPAH and non-PAH patients. B) Determine the effect of HMGB1 on PAEC and PASMC proliferation, migration, and apoptosis. C) Determine the effect of HMGB1 on PAEC activation. D) Use siRNA to determine a specific role for individual PRRs in mediating the effects of HMGB1 on PAEC and PASMC. These data will provide key mechanistic evidence of a role for HMGB1 and PRRs in promoting vascular remodeling and endothelial activation in PAH. The proposed research is based on a foundation of strong preliminary data. It is designed to extend our preliminary findings in animals to humans and further investigate the role of DAMPs and PRRs as key mediators of the innate immune response in PAH. Upon completion of the proposed research we will have a stronger foundation upon which to 1) further investigate the role innate immunity in PAH and 2) identify novel therapeutic targets related to innate immunity for the treatment of PAH.